F-actin cleavage in apoptotic outer hair cells in chinchilla cochleas exposed to intense noise

Sensory transduction in the cochlea and the vestibular labyrinth depends on the cycling of K+. In the cochlea, endolymphatic K+ flows into the sensory hair cells via the apical transduction channel and is released from the hair cells into perilymph via basolateral K+ channels including KCNQ4. K+ may be taken up by fibrocytes in the spiral ligament and transported from cell to cell via gap junctions into strial intermediate cells. Gap junctions may include GJB2, GJB3 and GJB6. K+ is released from the intermediate cells into the intrastrial space via the KCNJ10 K+ channel that generates the endocochlear potential. From the intrastrial space, K+ is taken up across the basolateral membrane of strial marginal cells via the Na+/2Cl-/K+ cotransporter SLC12A2 and the Na+/K+-ATPase ATP1A1/ATP1B2. Strial marginal cells secrete K+ across the apical membrane into endolymph via the K+ channel KCNQ1/KCNE1, which concludes the cochlear cycle. A similar K+ cycle exists in the vestibular labyrinth. Endolymphatic K+ flows into the sensory hair cells via the apical transduction channel and is released from the hair cells via basolateral K+ channels including KCNQ4. Fibrocytes connected by gap junctions including GJB2 may be involved in delivering K+ to vestibular dark cells. Extracellular K+ is taken up into vestibular dark cells via SLC12A2 and ATP1A1/ATP1B2 and released into endolymph via KCNQ1/KCNE1, which concludes the vestibular cycle. The importance of K+ cycling is underscored by the fact that mutations of KCNQ1, KCNE1, KCNQ4, GJB2, GJB3 and GJB6 lead to deafness in humans and that null mutations of KCNQ1, KCNE1, KCNJ10 and SLC12A2 lead to deafness in mouse models.     

K(+) cycling and its regulation in the cochlea and the vestibular labyrinth

Potassium (K(+)) plays a very important role in the cochlea. K(+) is the major cation in endolymph and the charge carrier for sensory transduction and the generation of the endocochlear potential. The importance of K(+) handling in the cochlea is marked by the discovery of several forms of hereditary deafness that are due to mutations of K(+) channels. Deafness results from mutations of KCNQ4, a K(+) channel in the sensory hair cells, as well as from mutations of the gap junction proteins GJB2, GJB3 and GJB6 that may facilitate cell-to-cell movements of K(+). Deafness results also from mutations of KCNQ1 or KCNE1, subunits of a K(+) channel that carries K(+) from strial marginal cells and vestibular dark cells into endolymph. Further, deafness results from mutations of KCNJ10, a K(+) channel that generates the endocochlear potential in conjunction with the high K(+) concentration in strial intermediate cells and the low K(+) concentration in the intrastrial fluid spaces. This review details recent advances in the understanding of K(+) transport and its regulation in the cochlea and the vestibular labyrinth.     

Reactive blue 2, an antagonist of rat P2Y4, increases K+ secretion in rat cochlea strial marginal cells

Extracellular ATP decreases K+ secretion in strial marginal cells via apical P2Y4 receptors. We investigated the effect of reactive blue 2 (RB-2), an antagonist of rat P2Y4, on rat strial marginal cells using a voltage-sensitive vibrating probe. The application of RB-2 increased K+ secretion in a dose-dependent manner, and this increase was characterized as a peak followed by a partial relaxation to a steady-state. Moreover, this response was similar to that caused by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Suramin had no similar effect, except at high concentration. Thus, we tested the effects of these chemicals on P2Y4 receptors in strial marginal cells. Both RB-2 and DIDS had antagonistic activities at P2Y4, and the antagonist potency at P2Y4 paralleled the potency of K+ secretion. Interestingly, 2'- and 3'-O-(4-benzoyl-benzoyl)adenosine 5'-triphosphate (BzATP) exhibited an agonistic effect at P2Y4 receptor, which was blocked by RB-2, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Based on these results, we speculate that direct and/or indirect inhibitory mechanisms between P2Y4 and KENQ1/KCNE1 K+ channels exist in strial marginal cell.     

KCNQ1/KCNE1 potassium channels in mammalian vestibular dark cells

The high [K(+)] in the inner ear endolymph is essential for mechanosensory transduction in hearing and balance. Several ion channels, including a slowly activating, voltage-dependent, outwardly conducting K(+) channel composed of the KCNQ1 (KvLQT1) and KCNE1 (IsK/minK) subunits, are expressed at the apical surface of vestibular dark cells. We investigated the underlying molecular mechanisms of this conductance using in situ hybridization, RT-PCR, and immunocytochemistry and by tracking the ultrastructural changes of vestibular structures in kcne1(-/-) mice. In the wild type mice, the KCNE1 and KCNQ1 proteins are expressed specifically at the apical membrane of dark cells, as early as gestational day (GD) 17 for KCNE1 while KCNQ1 mRNAs can be detected at GD 18. This is the first demonstration that the two protein components of this potassium channel co-localize in a polarized fashion at the cellular level. Although the vestibular end-organs are normal at birth in kcne1(-/-) mice, they begin to show modifications during postnatal development: we observed an increase in the height of the dark cells, in their number of mitochondria, and in basolateral membrane infoldings. Subsequently, the epithelium degenerates and the endolymphatic space collapses. Similar changes are known to occur in the cardio-auditory Jervell--Lange-Nielsen syndrome which is caused by mutations in the same channel.     

Inner ear abnormalities in a Kcnq1 (Kvlqt1) knockout mouse: a model of Jervell and Lange-Nielsen syndrome.

HYPOTHESIS: Mice lacking functional KCNQ1 (previously known as KvLQT1) channels exhibit functional and structural abnormalities that indicate disturbed production of endolymph. BACKGROUND: Congenital deafness associated with cardiac conduction abnormalities (Jervell and Lange-Nielsen syndrome) is associated with dysfunctional KCNQ1/KCNE1 channel complex. This potassium channel plays a critical role in the production and homeostasis of endolymph by the stria vascularis. A preliminary report documented severe abnormalities of the scala media and vestibular compartments of a single mouse lacking functional KCNQ1 alleles. METHODS: Hearing thresholds were measured in three Kcnq1 knockout mice, two heterozygous mice, and one wild-type mouse by auditory brainstem response recordings using clicks, after which the temporal bones were removed. After fixation and dehydration, the ears were embedded in araldite, sectioned at 20-microm thickness, stained with toluidine blue on glass slides, and examined with the light microscope. RESULTS: Kcnq1 knockout mice were deaf and demonstrated circling behavior. They exhibited a marked atrophy of the stria vascularis, contraction of the endolymphatic compartments, and collapse and adhesion of surrounding membranes. There was a complete degeneration of the organ of Corti and an associated degeneration of the spiral ganglion. CONCLUSION: Kcnq1 knockout mice exhibit histopathologic findings that are comparable to those reported in human temporal bone cases of Jervell and Lange-Nielsen syndrome, and provide further evidence that KCNQ1 channel dysfunction can lead to congenital deafness in this syndrome.     

Ultracytochemical study of Ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the inner ear of the squirrel monkey

The localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of the Na(+),K(+)-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K(+)-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K(+)-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.     

Quantitative assessment of the rat stria vascularis

Stria vascularis tissues from standardized regions in the basal, middle and apical turns of the rat cochlear duct were assessed quantitatively. Strial width, number of marginal cells across the strial width, radial area, as well as the volume density of the different components of the stria vascularis were determined for each standardized region. Strial width, number of marginal cells across the strial width and the radial area were greatest in the basal region and least in the apical region of the cochlea. The volume density of intermediate cells and capillary space was statistically unchanged in the three examined regions of the stria vascularis. However, the volume density of marginal cells and that of basal cells were different between regions. The volume density of marginal cells was highest in the basal turn while the volume density of basal cells was greatest in the apical turn. An objective assessment of the response of the stria vascularis to environmental conditions can be made by kant of its cellular architecture, providing a means to compare the effects of various agents between animal models used to study human inner ear dysfunction.     

Modulation of the rat stria vascularis in the absence of circulating adrenocorticosteroids

Structural changes in the cellular morphology of the rat stria vascularis from a standardized region of the basal region and from a standardized region in the apical region of the rat cochlear duct were measured using stereological methods after removal of endogenous levels of adrenal steroids by bilateral adrenalectomy. Although there were some inconsistent and insignificant alterations in the volume density of intermediate and basal cells, a decreased volume density of marginal cells in both the basal region and in the apical region in adrenalectomized (ADX) animals as compared to sham animals was consistent with a concomitant significant increased (p less than or equal to 0.05) volume density of intercellular space as observed in both the basal and apical regions of the stria vascularis of ADX animals. Findings of this study indicate that the strial cells of the stria vascularis react differently and independently in response to the removal of adrenal steroids, and such strial responses occur uniform in both the base and apex.     

耳蜗血管纹组织块的缘细胞培养

目的 :建立豚鼠耳蜗血管纹 (SV)组织块缘细胞 (MCs)的培养方法 ,为进一步研究药物耳毒性及其作用机制奠定基础。方法 :2 6只豚鼠按SV培养时间随机分成 4组 :2 4h组 (n =8) ;72h组 (n =8) ;>72h组 (n =8) ;对照组 (新鲜SV固定组 ,n =2 )。显微解剖数段连同螺旋韧带的SV组织块 ,置于 5 %CO2 / 95 %空气的二氧化碳恒温 (37℃ )培养箱中进行培养 ,分别进行形态学和组织学观察。结果 :培养 2 4hSV组织块保持良好活性 ,其组织学结构与新鲜固定的SV结构无明显差异 ;培养 72hSV组织块与新鲜固定的SV在组织学结构方面有显著性差异 ,不能观察到正常的SV结构 ,组织结构松散 ,缘细胞从组织块离心性生长出来 ;从SV组织块培养出的缘细胞能在培养皿内存活 13d。结论 :采用组织块培养技术 ,成功地建立了豚鼠耳蜗SV组织块的缘细胞培养方法 ;培养 2 4h的SV组织块光镜下保持了良好活性和正常组织学结构 ,可用来进一步研究药物耳毒性及其作用机制。     

Luminal non-specific cationic channels in cultured strial marginal cells of guinea pig and gerbil as determined by patch clamp technique.

Using primary cultures of marginal cells of stria vascularis from guinea pig and gerbil, ionic channels located on the luminal membrane were investigated by means of patch clamp technique. Recordings were performed in cell-attached and inside-out configurations. In cell-attached configuration, single channel activity was identified with a conductance of about 25 pS. I-V curve was linear. The probability of opening was increased upon depolarization. Up to 7 channels could be present in the same patch, indicating a rather high density. In inside-out configuration, the reversal potential was 0 mV, suggesting a non-specific cationic channel. These luminal non-specific cationic channels would allow the passive K+ efflux and Na+ influx across the apical membrane of marginal cells. This finding is consistent with the "one-pump" model of strial activity. The present study suggests that culture of strial marginal cells may be a suitable model for in-depth investigation of endolymph physiology.     

Estrogen acutely inhibits ion transport by isolated stria vascularis

We investigated the nongenomic effects of female sex steroid hormones on the short circuit current (I(sc,probe)) across gerbil stria vascularis using the voltage-sensitive vibrating probe. The strial marginal cell epithelial layer produces I(sc,probe) by secreting K+ via I(Ks) channels in the apical membrane. Application of 17beta-estradiol (E2) caused a decrease of I(sc,probe) in a dose-dependent manner (10 nM-10 microM) within seconds. Tamoxifen, a competitive inhibitor of the intracellular estrogen receptor, did not change the inhibitory effect of E2. Activation of I(Ks) channels by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in the presence and absence of E2 was used to test the mechanism of action. The results were consistent with a direct inhibitory effect of E2 on the I(Ks) channels. By contrast, progesterone caused a transient increase of I(sc,probe). These results suggest that E2 decreases secretion of K+ by inhibition of I(Ks) channels via a nongenomic mechanism at concentrations near those occurring under some physiologic conditions while progesterone caused only transient effects on I(sc,probe).     

The volume density of cells and capillaries of the normal stria vascularis

The purpose of this study was to provide morphometric (i.e. quantitative anatomical) data on the normal chinchilla stria vascularis. Five normal chinchillas were used in the present investigation and four regions of the cochlea were examined in each animal. The width, radial area and number of marginal cells across the stria's width increased from the cochlear apex toward the base. The increase in strial width and area appeared to be due to hyperplasia of the marginal cells. The mean total endolymphatic surface area of the stria vascularis was estimated to be 7.4 mm2 (S.E. = 1.23). The mean total volume of the stria vascularis was estimated to be 0.15 microliter (S.E. = 0.01). In addition, using a stereological method we found that the volume density of the cells and capillaries of the stria vascularis was constant along the length of the scala media. The mean (+/- S.E.) volume density of the stria cells and capillaries was estimated to be: marginal cells = 0.528 (0.013), intermediate cells = 0.212 (0.026), basal cells = 0.163 (0.009) and capillaries = 0.097 (0.009).     

Immunoreactivity of endothelins and endothelin receptor in the stria vascularis of the mouse cochlea

Immunoreactivities of endothelin-1, endothelin-3, endothelin receptor type A, and Na,K-ATPase were investigated in the stria vascularis of adult male WBB6F1 +/+ mice and in that of W/Wv mutants lacking strial intermediate cells. In the +/+ mice, electron microscopic immunoreactivity for the endothelins was seen on the rough endoplasmic reticulum, Golgi apparatus, cytoplasmic vesicles and lysosomes exclusively in the strial intermediate cells by the postembedment method. Immunoreactive endothelin receptor A was localized along the plasma membrane of strial marginal cells of both wild and mutant types although the immunoreactivity of the latter was much less than that of the former by the preembedment method. These findings suggest that the endothelins, which are produced in the strial intermediate cells, may play a role in the maintenance of the stria vascularis function in the +/+ mice. Since the plasma membrane of the marginal cells of the W/Wv mice, which do not generate a high positive endocochlear potential, also showed immunoreactivity for Na,K-ATPase, it seems likely that the endothelins are involved in the activation of sodium pump of the strial marginal cells by mediation of endothelin receptor A. In addition, the role of lysosomes in the crinophagy of the endothelins in the strial intermediate cells is proposed in the +/+ mice.     

Migration of cochlear lateral wall cells

The role of apoptosis and proliferation in maintenance of cochlear lateral wall cells was examined. The methods employed for detection of apoptosis were the Hoechst fluorescence stain and TUNEL (TdT-mediated dUTP-biotin nick-end-labeling) assay, and proliferations were 5-bromo-2'-deoxyuridine (BrdU) incorporation and presence of the proliferating cell nuclear antigen. The incidence of apoptosis in the strial marginal cell was 50% greater (32.9+/-3.7%) than strial intermediate and basal cells but similar to spiral ligament cells. Although division of marginal strial cells was rarely detected, a significant number of proliferating cells in the remaining stria vascularis and spiral ligament were observed. These data implied that replacement of marginal cells arose elsewhere and could be followed by a BrdU-deoxythymidine pulse-chase study. At 2 h post injection, nuclear BrdU in marginal cells was not detected; however, by 24 h post injection, 20-25% of marginal cell nuclei were BrdU-positive. These observations are consistent with the hypothesis that marginal cells were replaced by underlying cells. Cell migration appears to be an important mechanism for preserving the function and structure of the stria vascularis.     

Ultracytochemical study of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the inner ear of the squirrel monkey

The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of the Na+, K+-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K+-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K+-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.     

Alterations in the stria vascularis in relation to cisplatin ototoxicity and recovery

We have investigated whether or not cisplatin-induced depression of the endocochlear potential (EP), and its subsequent recovery, possesses a morphological correlate in the stria vascularis. Guinea pigs implanted with round window electrodes were treated daily with cisplatin (1.5 mg/kg/day) until the compound action potential showed a profound hearing loss (> or =40 dB at 8 kHz after 5-18 days). Animals were either sacrificed immediately after the shift in hearing threshold ('SHORT' group) or allowed to recover for > or =4 weeks and subsequently sacrificed ('LONG' group). Control animals ('CONTROL' group) were not treated with cisplatin. Using stereological methods we measured the total strial cross-sectional area together with the areas occupied by the different strial components: the marginal, intermediate and basal cells. The total strial cross-sectional area in the basal turn of the LONG group was found to be significantly smaller than that of the SHORT and the CONTROL groups, whereas the EP was normal in the LONG group (in comparison to the CONTROL group) and markedly decreased in the SHORT group. The smaller area in the LONG group was mainly due to a decrease in the area occupied by the intermediate cells and to a lesser extent to a decrease in the marginal cell area. The area occupied by the basal cells did not change. Thus, the marked decrease in EP after 5-18 days of cisplatin administration was not related to shrinkage of the stria vascularis. Moreover, 4 weeks later the EP showed full recovery, whereas the stria vascularis had shrunk markedly.     

Analysis of structural changes in the stria vascularis following chronic gentamicin treatment

Changes in the stria vascularis following chronic gentamicin treatment have been examined using quantitative methods. Albino guinea pigs were given gentamicin at 100 mg.kg-1.day-1 subcutaneously for 10 days. Comparisons were made of strial tissue from the treated animals sacrificed either 1 h or 4 weeks following the last injection with that from saline-injected controls. Strial width (spiral prominence to Reissner's membrane) and marginal cell (MC) number across the stria were determined from scanning electron micrographs. Strial thickness (endolymphatic surface to spiral ligament) and the volume fractions of the strial components (MCs, intermediate cells (ICs), basal cells (BCs) and capillaries) were derived from thin sections. Qualitative changes to both MCs and ICs were apparent 1 h after the last injection. At four weeks post-treatment, there was a small, but statistically significant, decrease in the number of marginal cells and a highly significant decrease in strial thickness. This was almost entirely due to a highly significant decrease in the volume fraction of MCs (i.e. shrinkage). The volume fraction of ICs was increased but this could be accounted for by MC shrinkage; after allowing for the reduction in strial thickness, the volume occupied by ICs was unchanged. Thus, following chronic gentamicin treatment, the stria is affected but significant progressive and permanent structural effects are confined to the marginal cells.     

Immunolocalization of ClC-K chloride channel in strial marginal cells and vestibular dark cells

Secretion of K⁺ into endolymph depends on a particular constellation of ion transport proteins in the apical and basolateral membranes of strial marginal cells and vestibular dark cells. One fundamental component is the large chloride conductance of the basolateral membrane, which recycles chloride taken up by the Na⁺-K⁺-Cl⁻ cotransporter in the same membrane. Evidence has been reported recently that supports ClC-K, a channel subunit previously thought to be specific to the kidney, as being the molecular entity underlying this conductance. We have isolated protein from the gerbil kidney, stria vascularis and vestibular labyrinth and found by Western blot analysis a 60 kDa band, a 48 kDa band and 54 and 70 kDa bands, respectively, specifically labeled by ClC-K antibody. Subsequent immunohistochemical observations of the inner ear tissues with a confocal microscope on fluorescently labeled tissue sections showed the staining to be restricted to the basolateral region of strial marginal cells and vestibular dark cells. The cochlear staining was distinct from the distribution of the Kir4.1 (KCNJ10) K⁺ channel, known to be present only in strial intermediate cells. These findings support the contention that ClC-K is an important component of the basolateral Cl⁻ conductance that participates in K⁺ secretion by these epithelia.